In the United States, hepatitis C virus (HCV) is the major etiologic agent of chronic viral hepatitis. The majority of infected individuals will develop progressive liver disease, which may include cirrhosis and hepatocellular carcinoma. Research on HCV has been severely hampered by the lack of an in vitro cell culture system and an adequate animal model. As a result, effective therapies against HCV have yet to be developed. Treatment with interferon is currently the only available therapy for HCV infection. Unfortunately, less than 50 percent of patients treated with high doses of interferon show a sustained response with eradication of HCV. A possible genetic basis for interferon resistance has been identified in a small, highly conserved domain of the NS5A gene product of HCV-1b, termed the interferon sensitivity determining region (ISDR). It is thought that NS5A may mediate HCV resistance to interferon by interacting with one or more interferon-induced cellular proteins associated with the interferon antiviral response. The P.I. laboratory has demonstrated that NS5A directly interacts with the interferon-induced proteins kinase, PKR, a primary mediator of the antiviral effects of interferon. Significantly, the ISDR appears to be required for this interaction. It is their hypothesis that NS5A is responsible for mediating HCV resistance to interferon therapy, at least in part, through a direct interaction with PKR. To test this hypothesis, they will use a multifaceted approach to characterize and define the interaction of NS5A with PKR. Specific goals of this proposal include: (1) an in- depth structure-function analysis of the NS5A-PKR interaction. NS5A deletion mutants will be constructed and site-specific mutations introduced into NS5A using mutD5 mutagenesis. Mutant NS5A constructs will be examined using in vitro and in vivo assays for PKR activity as well as for ability to reverse the PKR-induced slow-growth phenotype in yeast. (2) Establishment of stable NS5A-expressing Huh- 7 cell lines to examine the effects of NS5A on PKR function and activity and to obtain information regarding, the biological role of NS5A in HCV-infected cells. Stable NS5A-expressing NIH 3T3 cell lines will also be constructed to examine the potential role of NS5A in transformation and tumorigenesis. (3) Identification of NS5A phosphorylation sites in vivo. The importance of such sites for NS5A function will be examined by site-directed mutagenesis.